4: Innate Immune Response to S. aureus Biofilm PI: Bayles, K.W. SPECIFIC AIMS Biofilms are adherent communities of bacteria contained within a complex matrix. From a clinical standpoint, biofilm infections on body surfaces or medical devices represent a serious therapeufic challenge since organisms are recalcitrant to conventional antibiotics. Although host immune responses to planktonic bacteria have been relafively well characterized (34, 35, 70, 108, 112), little information is currently available regarding host immunity to S. aureus biofilms and how organisms modulate anti-bacterial effector mechanisms when organized in this protective milieu. To date, the majority of studies invesfigafing the innate immune response to biofilms have been performed with P. aeruginosa and S. epidermidis (20, 51, 52, 73, 85, 114), where neutrophils (PMNs) have been shown to phagocytize biofilm-associated bacteria and produce oxidative bursts, albeit at reduced levels compared to planktonic bacteria. To the best of our knowledge, only one publicafion exists where leukocyte responses to S. aureus biofilm have been direcfiy examined (74). This study demonstrated that leukocytes are capable of infiltrating S. aureus biofilms; however, the authors utilized a mixed population of peripheral blood leukocytes and as such, definifive conclusions regarding the cell type(s) interacting with the biofilm cannot be made. Therefore, additional mechanistic studies are needed to advance our understanding of the cross-talk between S. aureus biofilm and host innate immunity. The hypothesis ot this proposal is that S. aureus biofllm skews the host innate immune response from a classical pro-inflammatory bactericidal phenotype towards an anti-inflammatory, pro-flbrotic response to favor bacterial persistence. We will focus on the funcfional importance of key bacterial recognifion molecules (Toll like receptor 2 (TLR2) and TLR9) as well as anti-microbial mediators (INOS) by studying the pathogenesis of S. aureus biofilm using a foreign body infection model (17, 101) with mice deficient for these various molecules. We will also investigate the effects of purified PMN, macrophages (MO), and dendritic cells (DC) on biofllm survival and whether these immune cells induce changes in biofllm gene expression by transcriptional profiling. In addifion, we will employ in vivo bioluminescence imaging (IVIS) to monitor the immune response throughout biofilm development utilizing reporter mouse strains engineered to express luciferase under the control of promoters pivotal in host immunity to gram-posifive bacteria (i.e. iNOS, TLR2, and NF-KB). Finally, we will examine the host immune response to S. aureus murein hydrolase {cidA and IrgAB), nuclease {nuc), and nitric oxide reductase {nor) mutants from Projects 1 and 3 of this PPG in the mouse foreign body infection model. To address these objectives, the following specific aims will be performed. 1. Compare host innate Immune responses to S. aureus biofilm versus planktonic infection. These studies will 1) define the interactions between phagocytes and S. aureus in vitro when bacteria are grown in biofilm versus planktonic culture; 2) monitor changes in gene expression in S. aureus biofilms following exposure to innate immune cell populafions using transcripfional profiling; 3) characterize innate immune infiltrates into S. aureus biofilm in vivo and their activation profiles; and 4) examine the temporal activation of NF-KB, a major transcriptional regulator for a wide-array of inflammatory genes, during S. aureus biofilm growth using IVIS. 2. Define the role of extracellular bacterial DNA (eDNA) and peptidoglycan (PGN) in modulating host innate immunity to S. aureus biofllm. These experiments will 1) evaluate the host response to S. aureus biofilms using mice deflcient for TLR9 (receptor for bacterial DNA) and TLR2 (receptor for PGN); 2) deflne the kinefics of TLR2 activafion during biofilm development using TLR2-luciferase reporter mice and IVIS; and 3) evaluate the ability of S. aureus nuclease {nuc) and murein hydrolase {cidA, IrgAB) mutants to form biofilms in TLR9 and TLR2 KO mice, respecfively. 3. Examine the immune mechanisms leading to fibrotic encapsulation of S. aureus biofilms. These studies will 1) investigate whether S. aureus biofilm programs a host fibrofic response by facilitating the transition of MO from a classically (Ml) to an alternatively (M2) activated phenotype; 2) examine the funcfional importance of INOS in regulating biofilm encapsulation; and 3) track the inflammatory response to a S. aureus mutant engineered to lack an enzyme involved in bacterial NO metabolism {nor) and the role of host-derived NO in this process. 149